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Original Article
Email: nknemrawi@just.edu.jo
Received: 31 Mar 2018, Revised and Accepted: 23 Jun 2018
ABSTRACT
Objective: The aim of this work was to prepare chitosan nanoparticles (CS NPs) using sodium tripolyphosphate (TPP) as crosslinker and to study
the effect of chitosan polymeric properties and experimental conditions on the properties and stability of NPs.
Methods: CS NPs were prepared by ionic gelation method, using TPP as a crosslinker. The particle size (PS), polydispersity index (PDI), zeta potential
(ZP) and the morphologies of the NPs were studied. CS NPs prepared by varying the concentration of TPP, Chitosan molecular weight and its degree of
deacetylation, the stirring speed, the rate of TPP addition and the freeze-drying method to study the effect of these variables on the NPs. The stability of
the CS NPs was evaluated by storing aqueous suspensions of NPs and comparing the PS, PDI and ZP at the beginning and the end of the experiment.
Results: This study shows that the PS, ZP and dispersity of the NPs depend on the chitosan polymeric properties and experimental conditions. The
NPs sizes range between 145.73 and 724.23 nm. They all carried positive charges ranging between+4.32 and+43.67 mV. Most of the NPs have the
same sizes after freeze-drying, but showed higher monodispersity and ZP, indicating higher stability. After twenty days of studying the stability, the
NPs that had low ZP showed a large increment in size in comparison to the highly charged NPs.
Conclusion: In conclusion, the polymeric properties and formulation variables in the ionic gelation method have a great influence on the CS NPs formed.
Keywords: Chitosan, TPP, Stability, Nanoparticles
© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2018v10i5.26375
Sigma-Aldrich (St. Louis, MO). Sodium tripolyphosphate (TPP) was order to investigate its effect on the physicochemical properties of
purchased from AZchm (china). All other chemicals and reagents the NPs. TPP concentration, stirring speed, molecular weight of CS,
used were of analytical grade. CS degree of deacetylation and rate of adding TPP were investigated.
Methods Characterization of CS NPs
Preparation of different molecular weights of chitosan The mean particle size (PS), polydispersity index (PDI) and zeta
potential (ZP) of the nanoparticles were measured using a
Two molecular weights of chitosan were used in this work, the first Zetasizernano ZS90 instrument (Malvern, UK). The particle sizes and
one was purchased from sigma and has a molecular weight of 50 PDI were determined at 25℃C using dynamic light scattering (DLS).
kDa. The other one was prepared in our laboratory by acid The zeta potential was calculated from the electrophoretic mobility
depolymerization and has a molecular weight of 15 kDa [21]. This of the NPs in aqueous solution using the Helmholtz–Smoluchowski
polymer was a result of cleaving high molecular weight chitosan of equation under an electrical field of 40 V/cm. All measurements
250 kDa. Hight molecular chitosan was dissolved in 2 M HCl to make were carried out in triplicate (n = 3).
1% solution. Then the solution was refluxed for two hours. After
that, the solution was added to ethanol in a ratio of 1:2 v/v and The morphologies of all CS nanoparticles were investigated using
cooled down. Finally, the precipitate was filtered and washed SEM (Thermo scientific, Germany). The samples were coated with
several times with ethanol and centrifuged at 4000 rpm for 5 min carbon film prior to analysis, and then, studied under a microscope.
(Thermo scientific, Germany). The obtained chitosan was freeze
dried for 24 h (Telstar, Spain) and the powder was kept in an The stability of the CS nanoparticles was evaluated by storing
amber-airtight glass bottle and stored at room temperature. aqueous suspensions of different formulas at room temperature
(~25 °C). Few milligrams of each NP formulation were dispersed in
Preparation of different DDA of chitosan deionized water in a transparent glass bottle and observed for
twenty days. The particle size, PDI, and the zeta potential were
Three different degrees of deacetylation (DDA) were used to prepare measured at the beginning and the end of the experiment.
CS NPs in order to study the effect of chitosan DDA on the
physicochemical properties of the NPs. All three patches have the RESULTS AND DISCUSSION
same molecular weight, which is 15 kDa. The first one resulted from
the previous step and has a DDA ~92%. This polymer was further Preparation of different DDA and molecular weights of chitosan
used to prepare the other two DDAs by acetylation reaction. First, 1%
CS (15 kDa) was dissolved in water at pH of 6.5. Then the solution was The molecular weight and DDA of the chitosan purchased from
divided into two parts to which acetic anhydride was added to make sigma were determined and found to be 51.7±3.20 kDa and
1:0.15 and 1:0.6 molar ratios to prepare the 55% and 75% DDA, 75.01±1.34 %, respectively. This polymer molecular weight will be
respectively. The mixtures were stirred at 500 rpm at room referred to as 50 kDa and it’s DDA as 75% for simplicity. The
temperature and the pH of the solutions was maintained at 6.25. After chitosan that was prepared using high molecular weight chitosan
10 min, the chitosan solution was dialyzed against 4 L of distilled had a molecular weight of 15.32±0.24 kDa and a DDA of 91.9±0.22
water with gentle stirring at room temperature for 24 h using dialysis %. Further, the other two patches prepared using low molecular
tubes (12,000–14,000 Da molecular weight cut-off). Finally, the weight chitosan had DDA values of 75.09±1.37 % and 54.34±2.16 %.
dialyzed chitosan solution was poured in Petri dishes and dried The molecular weight of these polymers will be referred to as 15
overnight in an oven at 40 °C and the powder was transferred to an kDa and their DDAs as 90%, 75% and 50% for simplicity.
amber-airtight glass bottle and stored at room temperature [21].
Preparation of CS NPs
Characterization of chitosan molecular weight
Different patches of CS NPs were prepared by changing a single
The average molecular weight of chitosan was calculated by factor at each time. The effect of TPP concentration, stirring speed,
applying the Mark–Houwink’s equation [22]. Briefly, chitosan was the molecular weight of CS, CS DDA and rate of TPP addition were
dissolved in water and the viscosity was measured using a investigated and the mean particle size, PDI and zeta potential of the
viscometer (Vibro viscometer, Japan). Then, the viscosity average different formulations were measured (table 1). All NPs prepared in
molecular weight was calculated. this work showed spherical morphology confirmed by scanning
electron microscope (SEM) (fig. 1). Following is the effect of each
Characterization of chitosan DDA factor on nanoparticles physicochemical properties.
The DDA of the chitosan patches was determined using proton
nuclear magnetic resonance (1H-NMR) (Bruker Avance Ultra Shield
300 MHz, USA). Chitosan samples (30 mg/ml in D2O) were scanned
25 times. The DDA was calculated using the integrals of the H1
proton peak of the deacetylated monomer (H1-D) at 5.2 ppm and the
peak of the three protons of the acetyl group (H-Ac) at 2.8 ppm [23].
Preparation of CS NPs
The NPs were prepared using a modified ionic gelation method
previously described in the literature with some modifications.
Chitosan was dissolved in an aqueous solution of acetic acid (0.2
mg/ml) to form 0.5 mg/ml of chitosan solution. The chitosan
solution was stirred overnight at room temperature using a
magnetic stirrer at a pH of 3.6. After that, the chitosan solution
passed through a syringe filter (0.45 µm), and was then preheated in
a water bath at 60 ℃ for 10 min. In another beaker, TPP was
dissolved in HPLC water in different concentrations and then passed
through a syringe filter (0.45 µm) and cooled to 2-4 ℃. Finally, 5 ml
of TPP solution was added to 10 ml of chitosan solution and stirred Fig. 1: SEM image of CS NPs with a scale bar of 2 µm
for 10 min. The formed suspension was centrifuged (Thermo
scientific, Germany) for 30 min at 10000 rpm. The nanoparticles
were repeatedly washed with deionized water and freeze-dried for
48h at −80 °C to obtain the powdered nanoparticles [24, 25]. Effect of TPP Concentration on CS NPs
This procedure was repeatedly used to prepare CS nanoparticles, Different concentrations of TPP were used and their effects on the
and in each time, one of the preparation factors was changed in size, PDI and charge of the nanoparticles were investigated. As the
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amount of TPP used in the formulation increases, the size of the NPs reduce the mobility of chitosan and enhance its physicochemical
decreases to a minimum and then starts to increase again (fig. 2-A). properties such as stability. TPP is a polyanion that interacts with
In this study the amount of the TPP was increased while the amount the amino groups in chitosan to form a gel through inter-and
of chitosan and the volumes of the liquids were constant. In intra-molecular cross-linkages during the ionic gelation procedure
general, crosslinking agents are used in CS NPs formulation to [16].
Table 1: CS NPs, prepared under different conditions, mean particles size (nm), PDI and ZP (mV) before and after freeze-drying (n=3)
Before freeze drying After freeze drying
Formula TPP (mg) CS MWT* CS DDA TPP addition# Stirring+ PS (nm) PDI ZP (mV) PS (nm) PDI ZP (mV)
F1 7.5 50 90 0.25 700 208.50 0.08 17.37 169.00 0.20 20.33
F2 7.5 50 90 0.50 700 277.20 0.10 15.67 186.90 0.22 8.04
F3 7.5 50 90 1.00 700 247.13 0.15 18.13 229.80 0.27 6.06
F4 7.5 50 90 1.50 700 257.93 0.22 6.53 254.63 0.24 12.80
F5 7.5 15 90 2.50 700 388.07 0.41 8.14 346.13 0.50 13.73
F7 7.5 15 80 2.50 700 555.10 0.35 7.00 446.13 0.41 10.63
F6 7.5 15 55 2.50 700 663.07 0.51 4.78 582.07 0.52 4.32
F8 7.5 50 90 2.50 500 383.97 0.47 27.13 350.47 0.48 30.31
F9 7.5 50 90 2.50 1000 287.40 0.22 5.69 262.23 0.25 10.90
F10 7.5 50 90 2.50 1500 145.73 0.28 8.74 243.37 0.22 12.23
F11 1.5 50 90 2.50 700 610.00 0.54 40.00 576.13 0.54 43.67
F12 2.0 50 90 2.50 700 400.00 0.55 39.00 444.60 0.55 43.53
F13 3.0 50 90 2.50 700 356.70 0.28 30.50 295.87 0.12 35.30
F14 7.5 50 90 2.50 700 296.83 0.30 24.83 358.50 0.22 33.00
F15 15.0 50 90 2.50 700 508.43 0.28 5.38 540.80 0.21 8.16
F16 30.0 50 90 2.50 700 329.60 0.25 35.90 724.23 0.37 37.00
* CS molecular weight in kDa. #TPP addition rate in ml/min.+Stirring the rate in rpm. n=3 and data are given in mean±SD.
Fig. 2: The effect of different variables on CS NPs size (nm) before and after freeze-drying:(A) Effect of TPP amount (mg), (B) Effect of
speed of mixing (rpm), (C) Effect of chitosan DDA and (D) Effect of TPP addition rate(ml/min). n=3 and data are given in mean ± SD.
As the amount of TPP increases, the nanoparticle suspension This may be due to increased inter and intra cross-linking between
became progressively more turbid due to the formation of chitosan chitosan and TPP. On the other hand, as the amount of TPP continued
nanoparticles. When TPP amount was very low, the reaction to increase to more than 3 mg, CS NPs size started to increase. This
solution was clear and viscous like pure chitosan solution, which may be due to the fact that after chitosan molecules were fully
indicated that the TPP amount was not enough to form a crosslinked, the excess TPP would lead to more chitosan molecules
cross-linked structure of chitosan. As TPP amount increased to more being involved in the formation of a single nanoparticle [4, 26]. In
than 1.5 mg, the NPs started to be formed, and as the amount of TPP addition, as TPP concentration increases the samples become more
increased, the particle sizes decreased. monodispersed, and this is indicated by PDI values (fig. 3-A).
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Fig. 3: The effect of different variables on CS NPs PDI before and after freeze-drying:(A) Effect of TPP amount (mg), (B) Effect of speed of
mixing (rpm), (C) Effect of chitosan DDA and (D) Effect of TPP addition rate(ml/min). n=3 and data are given in mean ± SD.
In general, nanosuspensions having high positive charges above+10 The charges of the nanoparticles decrease as the TPP concentration
or below-10 are expected to have good stability while suspensions increases, which can be related to the neutralization of the
with charges between-10 and+10 are expected to have a higher protonated amino groups on chitosan by TPP anions (fig. 4-A). All
possibility of aggregation and less stability. All nanoparticles formulas prepared with different TPP amounts, except F15, had
prepared in this work carried positive charges. surface charges above+10, which indicates the high stability [27].
Fig. 4: The effect of different variables on CS NPs zeta potential (mV) beforeand after freeze-drying:(A) Effect of TPP amount (mg), (B)
Effect of speed of mixing (rpm), (C) Effect of chitosan DDA and (D) Effect of TPP addition rate(ml/min). n=3 and data are given in mean ±
SD.
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Fig. 5: The relationship between particles ZP (mV) and the particles size increment percentage. n=3 and data are given in mean±SD
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